YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa

Abstract

During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.

Klebsiella pneumoniae yfiRNB operon affects biofilm formation, polysaccharide production and drug susceptibility

“…The robust biofilms formed by K. pneumoniae yfiR cells are most probably due to enhanced polysaccharide production, triggered by local level c-di-GMP that can activate BcsA cellulose synthases. This is similar to what has been reported for both E. coli and P. aeruginosa, where homologues of yfiRNB have been shown to be involved in EPS production and biofilm formation (Malone et al, 2010;Raterman et al, 2013).…”

Importance of the Exopolysaccharide Matrix in Antimicrobial Tolerance of Pseudomonas aeruginosa Aggregates

“…Psl expression is also important for reduced phagocytosis (20). In line with this, increased immune evasion of ΔyfiR mutant strains has been reported, and it has been shown that disruption of pel and psl in a ΔyfiR mutant strain renders it as sensitive to phagocytosis by macrophages as the wild type (14).…”

“…Among the RSCV variants found in chronic lung infections are strains harboring loss-of-function mutations in wspF and yfiR, resulting in derepression of the di-guanylate cyclases WspR and YfiN, respectively. This in turn leads to increased production of the regulator molecule c-di-GMP and thereby increased production of biofilm matrix components, as well as a hyperadherent and autoaggregative phenotype (14,15). The occurrence of RSCVs has been correlated with increased risk of failure of antipseudomonal treatment (16).…”

Global Regulatory Pathways and Cross-talk ControlPseudomonas aeruginosaEnvironmental Lifestyle and Virulence Phenotype

“…As a strong link between c-di-GMP and the SCV phenotype exists, Malone and colleagues hypothesized that the SCV phenotype of the yfiR mutant was the result of upregulation of TpbB activity due to release of YfiR-mediated repression. The cellular concentration of c-di-GMP measured in the yfiR mutant displayed a significant increase in c-di-GMP when compare to wild type; this observation coupled with the results demonstrating DGC activity of purified TpbB (lacking the transmembrane domain) support the hypothesis that YfiR represses the DGC activity of TpbB (Malone, 2010). Epistasis experiments were performed to determine the regulatory interplay of YfiB, YfiR, and TpbB.…”

“…Three independent insertions were mapped to yfiR, the first gene of a predicted three-gene operon yfiBNR (Malone, 2010). YfiR is a periplasmic protein, previously identified as a suppressor of the tpbA mutant phenotype (Ueda, 2009a).…”